Set slide properly on the microscope and set new reference point

Click on Probes → Optical Fractionator Workflow → Start a new subject →

Enter cut thickness: 30 µm (cut with cryostat)

Enter interval according to the thickness: 5 if SNpc, 6 if STR

In Select Low Mag Lens, click 5X

Click on Next Step

Select the Contour of Interest(s)

In Select High Mag Lens select 40X

Click on Next Step

In Manually enter the average mounted thickness enter the corresponding one

Click on Next Step

In Define the Counting frame enter: 80 x 80 µm

Click on Next Step

Enter 25 Percentage

Click on Display Changes

Enter: 240x200 µm if SNpc, 620 x 490 µm if STR

Click on Display Changes

Click on Next Step

In Optical Disector Height enter: 5 µm, 20 µm

Click on Next Step

Select your first section

Click on Yes

Select the desired region

Click on start counting

Focus on the tissue

Click on OK

Select a number of markers according to the desired cell types

Count every defined region from every set section

Click on Next Step

Click on Display Probe Run List

Click on Section Name → Click on Counter/Marker Name → Select all sections belonging to the same region

Click on View Results

Click on the different markers to see the number of cells counted and the estimated cell number

Click on CE Scheaffer to view the area if needed

Repeat steps 17 through 20.1 for each region